electro blotted pvdf membranes  (Biosynth Carbosynth)

Journal: PloS one

Article Title: Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

doi: 10.1371/journal.pone.0005780

Figure Lengend Snippet: Figure 1. Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (PlaH) and light chain (PlaL) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS- PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 mg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37uC prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 mg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 mg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (161010) from log phase cultures (JEC21) were incubated 4 hr at 37uC in the presence (lane 1) or absence (lane 3) of 50 mg plasminogen and separated into cell wall and cytosol components, as described in Methods. Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 mg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37uC with 50 mg plasminogen (lanes 1–4, respectively). doi:10.1371/journal.pone.0005780.g001

Article Snippet: Western Blot analysis: Electro-blotted PVDF membranes were first incubated overnight at 4uC in PBS with 1.5% BSA and 0.07% Tween 20 then exposed for 1 hr (25uC) to rabbit anti-human plasminogen (Fitzgerald Industries), diluted 1:1000 in PBS+1.5% BSA.

Techniques: Incubation, Saline, Labeling, Extraction, SDS Page, Western Blot, Binding Assay

Journal: PloS one

Article Title: Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

doi: 10.1371/journal.pone.0005780

Figure Lengend Snippet: Figure 6. Identification of plasminogen-binding cell wall proteins of C. neoformans by 2D-PAGE and LC-MS/MS. Two-dimensional gel electrophoretic characterization of the cell wall proteome from a silver-stained gel (A), and the corresponding plasminogen-binding proteins (B) by ligand (plasminogen) overlay and western blot analysis with anti-plasminogen antibody. Indicated are the positions of identified plasminogen- binding proteins. Identified proteins shown in (B) are listed in Table S2. (* indicates spots sequenced following excision from the PVDF membrane due to lack of detection by silver-staining). doi:10.1371/journal.pone.0005780.g006

Article Snippet: Western Blot analysis: Electro-blotted PVDF membranes were first incubated overnight at 4uC in PBS with 1.5% BSA and 0.07% Tween 20 then exposed for 1 hr (25uC) to rabbit anti-human plasminogen (Fitzgerald Industries), diluted 1:1000 in PBS+1.5% BSA.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Two-Dimensional Gel Electrophoresis, Staining, Western Blot, Membrane, Silver Staining